A two-step bioconversion process for vanillin production from ferulic acid combining Aspergillus niger and Pycnoporus cinnabarinus

A two-step bioconversion process of ferulic acid to vanillin was elaborated combining two filamentous fungi, Aspergillus niger and Pycnoporus cinnabarinus. In the first step, A. niger transformed ferulic acid to vanillic acid and in the second step vanillic acid was reduced to vanillin by P. cinnabarinus. Ferulic acid metabolism by A. niger occurred essentially via the propenoic chain degradation to lead to vanillic acid, which was subsequently decarboxylated to methoxyhydroquinone. In 3-day-old cultures of P. cinnabarinus supplied with vanillic-acid-enriched culture medium from A. niger as precursor source, vanillin was successfully produced. In order to improve the yields of the process, sequential additions of precursors were performed. Vanillic acid production by A. niger from ferulic acid reached 920 mg l⁻¹ with a molar yield of 88% and vanillin production by P. cinnabarinus from vanillic acid attained 237 mg l ⁻¹ with a molar yield of 22%. However, the vanillic acid oxidative system producing methoxyhydroquinone was predominant in P. cinnabarinus cultures, which explained the relatively low level in vanillin.     

微生物转化方法生产香草酸与香草醛的初步研究

从实验室保藏的菌种中筛选到一株黑曲霉（Aspergillus niger）SW-33,能够将1g/L的阿魏酸底物转化为0.23g/L的香草酸,相应的摩尔转化率为29.35％;流加四次底物阿魏酸后,产物浓度达到1.11g/L,相应的摩尔转化率为44.9％。为了提高产物浓度,对培养基和发酵条件进行优化,使得该菌株能够将1g/l的阿魏酸底物转化为0.46g/L的香草酸,相应的摩尔转化率为57.81％。提取得到的香草酸,经HPLC测定,纯度为85.9％;提取收率为75.2％。用含香草酸的转化液,或者用提取的结晶香草酸,加入朱红密孔菌（Prcnporus cinnabarnus）SW-0203发酵培养液,可得到转化产物香草醛。     

Overproduction of laccase by a monokaryotic strain of Pycnoporus cinnabarinus using ethanol as inducer

AIMS: Laccase production by the monokaryotic strain Pycnoporus cinnabarinus ss3 was studied using ethanol as inducer in the culture medium. METHODS AND RESULTS: The effect of ethanol was tested at 10, 20, 30, 35 and 45 g l-1 and compared with that of ferulic acid, known until now as the most efficient inducer for laccase expression by P. cinnabarinus ss3. In the presence of 35 g l-1 ethanol, laccase activity (266 600 U l-1) and productivity (19 000 U l-1 day-1) were nine and fivefold higher compared with ferulic acid-induced cultures, and 155- and 65-fold higher compared with non-induced cultures, respectively. In vivo, ethanol added to the culture medium of P. cinnabarinus ss3 favoured a continuous and high expression of laccase gene. Under these conditions, P. cinnabarinus ss3 produced preferentially the isoenzyme LAC I. Ethanol added in vitro to the purified P. cinnabarinus ss3 laccase typically inhibited the enzymatic activity. CONCLUSIONS: In spite of an initial inhibitory effect on mycelial growth, ethanol was shown to be a very strong inducer for laccase expression by P. cinnabarinus ss3 allowing an average yield of 1-1.5 g l-1 laccase. SIGNIFICANCE AND IMPACT OF THE STUDY: This study identified P. cinnabarinus ss3 as an outstanding producer of laccase in the presence of ethanol as inducer. Ethanol is an inexpensive agricultural by-product and the process is simple to scale-up for industrial production.     

Laccase production by a monokaryotic strain of Pycnoporus cinnabarinus derived from a dikaryotic strain

Monokaryotic Pycnoporus cinnabarinus strains were obtained from the dikaryotic strain I-938. One of these, designated MK18, consistently produced high laccase activity. In cultures of MK18 and I-938 where ferulic acid was added as laccase inducer, laccase activity was enhanced about 2.5-fold reaching 3400 U/l for the MK18 strain. Laccase was purified to homogeneity and under the selected growth conditions, only one isoform of the enzyme was produced. The N-terminal sequence was similar to the amino terminal sequence of laccase II from Trametes versicolor. The enzyme was stable at 60 C for more than 1 h.     

黑曲霉电转化条件的研究

研究避免了繁琐的原生体制备过程,直接使用萌发的黑曲霉孢子进行电转化,以潮霉素B作为筛选标记,从孢子萌发时间、电场强度及质粒浓度等方面考察了电转化效率的影响因素。研究表明,针对A.nigerMGG029-ΔaamA,其理想的电转化条件:孢子龄为4d,孢子萌发时间为2h,电场强度为5kV/cm。在上述条件下分别使用1μg环状或线状pBC-Hygro质粒DNA进行转化,平均可以得到34个和51个转化子,而在同样条件下使用质粒pRS303H平均可以获得163个和258个转化子。     

利用固定化黑曲霉单宁酶制备没食子酸的研究

用海藻酸钙载体包埋单宁酶,制备出转化五倍子单宁成没食子酸能力较好的固定化酶。研究了固定化条件和固定化单宁酶的部分性质,结果表明：最佳固定化条件为海藻酸钠90mg包埋单宁酶546u(3mL,182u/Ml),在1%～2%CaCl₂中作硬化处理;固定化单宁酶的最适温度为45℃,在10～50℃范围内稳定;其最适Ph值为6.5,在Ph5～7之间基本稳定;在此基础上,进行了没食子酸实验室克量级酶法制备实验,3次实验没食子酸产品的平均产率达到61%。和目前所用工业生产没食子酸的硫酸水解法相当,具有潜在的工业开发价值。     

米曲霉和黑曲霉营养缺陷型的分离及原生质体的制备

米曲霉(Aspergillus oryzae)3042是目前国内酱油生产中广泛使用的菌种,而黑曲霉(Aspergillus niger)3350则是制醋业中广泛使用的菌种。前者具有较高的蛋白酶活性而后者具有较高的淀粉酶活性。在酱油生产中,为了提高原料利用率,改善酱油风味,希望获得一株既有较高的蛋白酶活性同时又具有较高淀粉酶活性的杂交菌株作为     

Biotransformation of glycyrrhizin by Aspergillus niger

Biotransformation of glycyrrhizin by Aspergillus niger was investigated and one new compound (1) and one known compound (2) were isolated and identified from the biotransformation products. These were 7β,15α-dihydroxy-3,11-dioxo-oleana-12-en-30-oic acid (1) and 15α-hydroxy-3,11-dione-oleana-12-en-30-oic acid (2). A biotransformation pathway was proposed from HPLC analyses at different reaction times. The biotransformation by A. niger included two stages: first, the two glucuronic acid residues at the C-3 position of glycyrrhizin were hydrolyzed to produce glycyrrhetic acid; and second, glycyrrhetic acid was oxidized and hydroxylated to compounds 1 and 2.     

Transformation of 2-hydroxydibenzofuran by laccases of the white rot fungi Trametes versicolor and Pycnoporus cinnabarinus and characterization of oligomerization products

Laccase, a ligninolytic enzyme, was secreted by each ofthe white rot fungi Trametes versicolor and Pycnoporus cinnabarinusduring growth in a nitrogen-rich medium under agitated conditions. Afteraddition of 2-hydroxydibenzofuran to cell-freesupernatants of the cultures, yellow precipitates wereformed. These precipitates were poorly soluble in waterand therefore readily separated from the supernatant. Theproducts formed were more hydrophobic than thesubstrate, as indicated by their longer retention times on areverse phase high-performance liquid chromatographycolumn. Mass spectrometric analysis of the purifiedproducts indicated the formation of oligomers. Analysis ofthe mixture of products by gas chromatography and massspectrometry after derivatization with diazomethanesuggested the formation of at least three dimeric and ninetrimeric products. Carbon-carbon and carbon-oxygenbonds were identified in the dimers and trimers,respectively. The nuclear magnetic resonance spectrum ofthe main dimer suggested coupling of the two monomersat the carbon one position.     

Optimization of Ellagitannase Production by Aspergillus niger GH1 by Solid-State Fermentation

Ellagic acid is one of the most bioactive antioxidants with important applications in pharmaceutical, cosmetic, and food industries. However, there are few biotechnological processes developed for its production, because it requires precursors (ellagitannins) and the corresponding biocatalyst (ellagitannase). The aim of this study was to optimize the culture conditions for ellagitannase production by Aspergillus niger in solid-state fermentation (SSF). The bioprocess was carried out into a column bioreactor packed with polyurethane foam impregnated with an ellagitannins solution as carbon source. Four strains of Aspergillus niger (PSH, GH1, HT4, and HC2) were evaluated for ellagitannase production. The study was performed in two experimental steps. A Plackett–Burman design was used to determine the influencing parameters on ellagitannase production. Ellagitannins concentration, KCl, and MgSO₄ were determined to be the most significant parameters. Box–Behnken design was used to define the interaction of the selected parameters. The highest enzyme value was obtained by A. niger PSH at concentrations of 7.5 g/L ellagitannins, 3.04 g/L KCl, and 0.76 g/L MgSO₄. The methodology followed here allowed increasing the ellagitannase activity 10 times over other researcher results (938.8 U/g ellagitannins). These results are significantly higher than those reported previously and represent an important contribution for the establishment of a new bioprocess for ellagic acid and ellagitannase production.